Comparison of Isolation Procedures for the Gas Chromatographic Determination of Methadone in Human Liver Sir: The use of methadone in the treatment of narcotic addicts (1-3) has increased the opportunities for the abuse of this drug ( 4 , 5 ) and hence the need for effective analytical procedures. Several techniques have been applied to the detection of methadone in biological fluids (&IO), but comparatively little information is available for comparable studies on tissues 111-14). Methadone has been isolated from tissue by acid ( 1 1 ) and alkaline digestion methods (12, 13) and by direct extraction (14). The alkaline digestion and the direct extraction methods gave similar recoveries, 78.7% (12) and 87% ( I d ) , respectively, from spiked samples. An improved analytical procedure utilizing the alkaline digestion gave a recovery of 85% ( 1 3 ) . This paper compares the extraction efficiency and cleanness of the alkaline digestion procedure developed for methadone analyses with that of the acid and ammonium sulfate (15) digestion methods commonly used for general toxicological analyses. The direct extraction method (14) was not used as it seemed to offer little advantage over the other methods and generally produces less pure extracts. Gas chromatography provided a simple, accurate, and reproducible method of analysis. EXPERIMENTAL Apparatus. Gas Chromatography (GC). The gas chromatograms were recorded on a Varian 600D (FID) fitted with either a 3% OV-17 glass or a 2.5% OV-1 stainless steel column, or on a Perkin Elmer F-11 (FID:l fitted with a 3%XE-60 glass column. The operating conditions were as follows: 3% OV-17 (2.5 m X 2-mm i.d.) on 60-80 mesh AW-DMCS Chromosorb G; a t 240 "C, 20 ml per minute of nitrogen carrier gas; 2.5% OV-1 (2.5 m X 2-mm i.d.) on 60-80 mesh .4W-DMCS Chromosorb G; at 220 "C, 20 ml per minute of nitrogen carrier gas; 3% XE-60 (1.5 m X 3-mm i.d.1 on 60-80 mesh DMCS Chromosorb W a t 190 "C; 20 ml per minute of nitrogen carrier gas. Thin Layer Chromatography (TLC). Thin layer chromatograms were obtained on Schleicher and Schuell F1500 LS 254 plates using methanol/ammonia (100:1.5) as the developing solvent. The compounds were detected by spraying with acidified iodoplatinate solution. Case History. A l6-year old male was admitted, unconscious, to a hospital. He died two days later without regaining consciousness. The youth had been remanded by the court several days earlier in connection with a drug charge. He was prescribed methadone treatment and it is thought that he saved the methadone and took an intravenous overdose. He died before his trial was scheduled by the court. Procedure. The fclllowing three methods were used for the isolation of methadone from the liver of the deceased. Alkaline Extractiom Liver (30 g) was digested with 30% potassium hydroxide (30 ml) as described by Wallace et al. (12).The solution was extracted three times a t pH 9-12 with an equal volume of hexane. Acid Extraction. Liver (50 g) was digested with concentrated hydrochloric acid (60 ml) and water (100 ml) as described by Nelson and Selkirk (11).'The digest was washed with ether and the pH adjusted to 9. The solution was then extracted three times with an equal volume of ether. Ammonium Sulfate Extraction, Liver (50 g) was digested with concentrated hydrochloric acid (10 ml) and water (40 ml), and the solution saturated with ammonium sulfate. The solution was then treated as described Iiy Jackson (15). Hexane (3 X 1 volume) and ether (3 X 1volume) were used for separate extractions a t p H 9. The smaller quanti-ty of liver used for the alkaline digestion was dictated in this case by insufficient sample. The extracts were dried with Analar sodium sulfate, evaporated to dryness, and taken up in 20 pl of chloroform. The concentrated extracts were then analyzed by thin layer chromatography and gas chromatography. The Rr's of the spots obtained on the thin layer chromato-
Table I. Gas Chromatographic Data a n d Levels of Methadone in Liver Samples GC column
3% OV-17
Retention time relative to trimipramine Estimated l e v e l s (pg/g) Acid digest Alkaline digest Amm. sulf. digest
2.50>
0.78 0.80 0.84
i:
+ Nil
0.05 0.02
01'-1
0.80 0.78 i 0.04 0.80 0.03 Nil
grams were compared with those of methadone and codeine. The gas chromatographic peaks were compared with those obtained for trimipramine and methadone. The methadone in the samples was quantitated by comparison of the GC peak height with that obtained for authentic methadone standards. All extraction procedures were conducted in duplicate.
RESULTS AND DISCUSSION The concentrated extracts were examined by gas chromatography on the 3% OV-17, 2.5% OV-1 and 3% XE-60 (retention time for methadone of 0.85 relative to trimipramine) columns and by thin layer chromatography ( R methadone relative to codeine of 1.26). Methadone could not be detected in either the hexane or ether extracts from the ammonium sulfate digestion. Impurities in the acid digest extract caused peak broadening on the XE-60 column and spot streaking on TLC and prevented satisfactory analysis for methadone by these methods. Conversely, the OV-17 and OV-1 columns gave good separation and allowed easy quantitation of the methadone in the acid digest extract. Methadone was identified in the alkaline digest extract on all three GC columns and also by TLC. The alkaline digest gave a much cleaner extract than the acid digest. This was undoubtedly due, a t least in part, to the use of hexane rather than ether as the extracting solvent. Ether was used in the acid digestion as it was decided to follow a standard toxicological screening procedure ( 1 1 ) . Nelson and Selkirk ( 1 1 ) developed a procedure for cleaning up the extract from the acid digestion where necessary by using the solubility of methadone hydrochloride in chloroform. As our acid digest extract could be readily analyzed on both OV-17 and OV-1, their clean-up procedure was considered unnecessary and was not used as it can result in a loss of a further 30% of the drug. The drug levels are listed in Table I. The results obtained on the two columns are in excellent agreement. Furthermore, excellent agreement was found for the quantitation by the two isolation procedures. Similar methadone recoveries of about 80% have been reported for the alkaline digestion procedures (12, 13) and the direct extraction procedure ( 1 4 ) . Furthermore, the acid digestion method gave identical recoveries to the direct extraction method (11). The equivalent methadone recoveries found in this work for the acid and alkaline approaches is therefore in agreement with the findings of these workers. The methadone level (0.8 Kg/g) in the liver is higher than the levels found in two of the four cases reported by Nelson and Selkirk ( 1 1 ) which, as in this case, did not involve other drugs but is low in comparison with other studies (14, 16).
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For the determination of methadone, gas chromatography was preferred to the oxidation procedures of Wallace et al. (12, 13).The latter methods involve the conversion of methadone to benzophenone, which is estimated spectrophotometrically, and suffer from a loss of specificity as several other drugs will produce benzophenone under the same conditions. A very minor peak (peak height less than 2% that of methadone) was detected in the gas chromatograms of both the acid and alkaline digest extracts at about R methadone of 1.50 on 3% OV-17 and 1.48 on 2.5% OV-1. This peak was tentatively identified as the primary methadone metabolite 1,5-dimethyl-3,3-diphenyl-2-ethylidenepyrrolidine which has been detected in biological specimens (13, 14). The low level present did not permit positive identification of the compound.
CONCLUSIONS Of the three isolation procedures studied, only the acid and alkaline extraction procedures were satisfactory for the determination of methadone in liver at the low levels frequently encountered in post-mortem investigations. If methadone is the only basic drug for which the tissue samples are being examined, then the isolation procedure of Wallace et al. ( 1 2 ) yields a cleaner extract and would be the method of choice. If other basic drugs are suspected, an acid digestion, as generally used in routine post-mortem analyses is quite satisfactory, but the detection of methadone by gas chromatography is limited by the choice of column. In the present study 3% OV-17 and 2.5% OV-1 columns were found to be more suitable than a 3% XE-60 column for acid digestion extracts.
LITERATURE CITED (1) (2) (3) (4)
V. P. Dole and M. Nyswander. J. Am. Med. Assoc., 193, 646 (1965). A. Goldstein and B. W. Brown Jr., J. Am. Med. Assoc., 214, 31 1 (1970). L. Lasagna, Pharm. Rev., 16, 47 (1964). R. Gardner, Lancet, 2, 650 (1970). (5) M. M. Baden, "Committee on Problems of Drug Dependence", National Academy of Sciences-National Research Council, Division of Medical Sciences, Washington, D.C.. 1970, pp 6767-9. (6) C. E. lnturrisi and K. Verebely, J. Chromatogr., 65, 361 (1972). (7) I. K. Ho, H. H. Loh, and E. L. Way, J. Chromatogr., 65, 577 (1972). (8) H. R. Sullivan and D. A. Blake, Res. Commun. Chem. fathol. Pharmacob, 3, 467 (1972). (9) J. Ramsey and D. B. Campbell, J. Chromatogr., 63, 303 (1971). (10) L. B. Hetland, D. A. Knowlton, and D. Couri, Clin. Chim. Acta, 36, 473 119721. - -, \
(11) P. E. Nelson and R . C. Selkirk, Chemistry Division, D.S.I.R., Auckland, N.Z., October 1974. unpublished results. (12) J. E. Wallace, H. E. Hamilton, J. T. Payte, and K. Blum, J. Pharm. Scb, 61, 1397 (1972). (13) H. E. Hamilton, J. E. Wallace, and K. Blum, J. Pharm. Sci., 63, 741 (1974). (14) A. E. Robinson and F. M. Williams, J. Pharm. Pharmacal., 23, 353 (1971). (15) J. V. Jackson, in "lsolatlon and Identification of Drugs,'' E. G. C. Clark, Ed., The Pharmaceutical Press, London, 1969. (16) A. E. Robinson in "Forensic Toxicology," Proceedings of a Symposium heid at the Chemical Defence Establishment Porton Down, 29-30 June 1972, B. Ballantyne, Ed., John Wright & Sons Ltd., Bristol, 1974.
Stuart J. Dickson Philippa A. Palmer Chemistry Division Department of Scientific and Industrial Research Private Bag Petone, New Zealand
RECEIVEDfor review December 5 , 1974. Accepted May 23, 1975.
I AIDS FOR ANALYTICAL CHEMISTS Break-Bulb Technique for Sample Introduction Frederick J. Tehan and James L. Dye' Department of Chemistry, Michigan State University, East Lansing, Mich. 48824
Handling of isolated amounts of materials, especially for use in vacuum systems has been a serious problem over the years ( I ) . In 1947, Coops et al. (2) introduced the use of fragile glass ampoules sealed to a ground-glass joint. Disadvantages included a total mass of approximately 2.5 grams, which prevented the use of very small samples, and the necessity to remove all grease from the ground glass joint. The general method was revised for the isolation of alkali metals in small quantities (approximately 50-100 mg) by Watt and Sowards ( 3 ) , and also by Shriver (1). These authors were able to eliminate the ground-glass joint and therefore reduce the bulb weight to 100-400 mg. However, the method could not be used for sealing-off small amounts of salts or liquids in the bulb without introducing air into the ampoules. Jones and Dewald ( 4 ) recently described a convenient technique for the preparation of sodium samples. I t is often desirable to obtain small quantities of materials that can be easily introduced into a vessel without the loss of vacuum or inert atmosphere. In this regard, the isoAuthor to whom correspondence should be addressed. 1876
lation of alkali metals in small quantities has posed especially serious problems. For example, if a 1 X 10-3M solution of sodium in 20 ml of ammonia is desired, less than 1 mg of alkali metal must be isolated. The accurate isolation of such a small mass of metal without oxide or other impurity is not trivial. An ideal sample-handling system should meet the following objectives: 1) Complete isolation of the sample from air and moisture. Both vacuum and inert atmosphere capabilities are desirable. 2) The ability to handle a wide range of sample weights. The method should be capable of delivering small as well as large amounts of material. 3) The ability to introduce samples in the form of liquids, solids, or solutions. 4) The preparation technique should be relatively rapid and simple to perform. 5 ) The method should allow quick handling and easy manipulation 'of the samples during an experiment. We have extended the general bulb technique to permit the isolation of very small known amounts of salts, solvents, and solutions. Premeasured milligram and sub-milligram quantities of alkali metals were also isolated in small, fragile, easily breakable glass ampoules. This method dif-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
